Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
  • C12Q1/701—Specific hybridization probes
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
  • A61B5/00—Measuring for diagnostic purposes; Identification of persons
  • A61B5/08—Detecting, measuring or recording devices for evaluating the respiratory organs
  • A61B5/097—Devices for facilitating collection of breath or for directing breath into or through measuring devices
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
  • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
  • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
  • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
  • B01L3/508—Containers for the purpose of retaining a material to be analysed, e.g. test tubes rigid containers not provided for above
  • B01L3/5082—Test tubes per se
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
  • B01L2200/02—Adapting objects or devices to another
  • B01L2200/025—Align devices or objects to ensure defined positions relative to each other
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
  • B01L2200/06—Fluid handling related problems
  • B01L2200/0684—Venting, avoiding backpressure, avoid gas bubbles
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
  • B01L2200/16—Reagents, handling or storing thereof
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L2300/00—Additional constructional details
  • B01L2300/04—Closures and closing means
  • B01L2300/041—Connecting closures to device or container
  • B01L2300/042—Caps; Plugs
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L2300/00—Additional constructional details
  • B01L2300/04—Closures and closing means
  • B01L2300/046—Function or devices integrated in the closure
  • B01L2300/047—Additional chamber, reservoir
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L2300/00—Additional constructional details
  • B01L2300/04—Closures and closing means
  • B01L2300/046—Function or devices integrated in the closure
  • B01L2300/048—Function or devices integrated in the closure enabling gas exchange, e.g. vents
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6844—Nucleic acid amplification reactions
  • C12Q1/6851—Quantitative amplification
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q2600/00—Oligonucleotides characterized by their use
  • C12Q2600/16—Primer sets for multiplex assays

Definitions

• This invention generally relates the measuring or testing processes involving enzymes, nucleic acids, or microorganisms; compositions or test papers therefor; processes of preparing such compositions; condition-responsive control in microbiological or enzymological processes; and to preparing nucleic acids for analysis, e.g., for polymerase chain reaction [PCR] assay.
• PCR polymerase chain reaction
• the COVID-19 virus (SARS-CoV-2) is transmitted through airborne particles in exhaled breath, causing severe respiratory disease.
• Diagnostic assays for active or prior infection rely on detecting viral RNA or antibodies to the virus. Diagnostic assays are usually performed on patient samples collected from a patient’s upper respiratory tract by saliva or nasopharyngeal (NP) swab. These sources have comparable sensitivities, with 97% agreement.
• the invention provides an alternative, effective RNA virus sample collection device.
• This sample collection device can replace nasopharyngeal swab, stabilization, RNA extraction, and reverse transcription with a rapid, single step.
• the collection device samples aerosolized particles from human breath.
• the invention provides a more direct and medically relevant sample for evaluating transmission risk than a nasopharyngeal swab.
• the invention provides a device called the
• BubblerTM which captures aerosolized RNA-containing particles from a subject’s breath.
• the device is used by having the subject be tested for RNA virus presence, bubbling a breath through an oil/aqueous solution/emulsion contained in the device.
• oil/aqueous solution/emulsion In the oil/aqueous solution/emulsion are reagents for carrying out an enzymatic reverse transcriptase (RT) reaction. See FIG. 1 (A).
• the enzyme activity then converts the viral RNA into stable, molecularly barcoded cDNA.
• the reverse transcriptase activity at the collection site advantageously enables the collection of samples compatible with downstream large-scale sequencing-based parallel diagnostics. See FIG. 3. Alternately, the sample can be diagnosed without sequencing, by PCR.
• the invention provides a method for reverse transcribing RNA from airborne SARs-CoV-2 viral particles into a sample-specific barcoded cDNA.
• the method comprises the step of first obtaining a deep breath sample from a patient, such as described in this specification concerning the use of the device of the invention. Next, the sample is reverse transcribed to viral cDNA using sample- specific barcoded primers. Optionally, the samples can then be pooled for analysis by a massively parallel assay.
• the invention provides the device for use as a screen for RNA virus (e.g., COVID-19, SARS, other coronaviruses, influenza viruses, rhinoviruses, or other RNA viruses) in human breath.
• RNA virus e.g., COVID-19, SARS, other coronaviruses, influenza viruses, rhinoviruses, or other RNA viruses
• the invention provides the device for use as a screen for RNA virus (e.g., COVID-19, SARS, other coronaviruses, influenza viruses, rhinoviruses, or other RNA viruses) in the environment by applying a vacuum pump to an air vent installed in hospital emergency rooms, airports, building heating, ventilation, and air conditioning (HVAC) air handling systems.
• RNA virus e.g., COVID-19, SARS, other coronaviruses, influenza viruses, rhinoviruses, or other RNA viruses
• the invention provides the device for use in detecting a DNA virus in exhaled breath.
• the invention provides the device for use in detecting nonviral nucleic acid in exhaled breath (e.g., DNA from tobacco; DNA from cannabis).
• the invention provides the device for use in detecting aerosolized DNA in the environment.
• the invention provides the device for use in collecting a sample that is used for sequencing to identify the strain of the virus.
• the invention provides the device for use in collecting a sample that is used for [massively parallel assay]
• the invention provides, the invention provides a device, wherein the receptacle is a balloon, such as a party balloon.
• RNA virus particles from human breath were readily precipitated from an inflated party balloon's interior surface after a one-hour incubation at -20°C.
• RT-PCR can readily detect rRNA in this liquid with no RNA extraction.
• the invention provides a rapid, high-throughput assay that advantageously enables large-scale survey sequencing. See FIG. 4.
• the BubblerTM could even be dispatched for home use, decreasing the current burden on clinical testing facilities.
• the invention provides a device and an improved method for determining infectivity. Without an ability to assay SARS-CoV-2 in human breath, a person having ordinary skill in the biomedical art cannot learn the time and circumstance (vaccinated, asymptomatic) when COVID-19 patients are infectious. This situation has been widely appreciated to have contributed to the public uncertainty during the 2020 COVID-19 pandemic.
• a diagnosis by sequencing can provide additional information such as viral load and strain identity.
• samples from barcode-enabled BubblersTM where the samples contain cDNA amplified using genetically barcoded primers, can be pooled and batch processed while retaining sample identity.
• the invention was tested in a clinical study that demonstrated the feasibility of molecular barcoding coupled with next-generation sequencing to quantitatively detect SARS-CoV-2 in a panel of human-constructed samples at a detection limit of 334 genomic copies/sample.
• FIG. 1 provides information about the BubblerTM device.
• FIG. 1(A) shows the product design. The person being tested exhales through a glass mouthpiece so that aerosolized particles containing viral and cellular RNAs are bubbled through a cool oil/aqueous emulsion. Aerosol particles condense in the aqueous phase and mix with a reverse transcription buffer which copies RNA into barcoded cDNA.
• FIG. 1(B) shows how a person being tested exhales gently into a hand-held BubblerTM for less than one minute to evacuate the lungs completely.
• FIG. 1(C) is a proof of concept. The cellular 18S rRNA was copied into DNA and amplified by PCR. The electrophoresis gel result shows that the BubblerTM isolates as much RNA in one breath as a conventionally-extracted RNA- labeled control, which was a ⁇ 2-hour Trizol reaction + reverse transcriptase.
• FIG. 2 is a diagram showing the massively parallel RNA virus diagnostic assay working on human-constructed samples.
• Each device contains a unique barcode appended to the reverse transcription (RT) primer (drawn in purple), adjacent to a random 3-mer(NNN), the reverse primer binding site (labeled Common primer), and a bacteriophage T7 promoter (T7) which is incorporated into the cDNA.
• the cDNA is treated to remove free primers and protein and then amplified via T7 in vitro transcription.
• the resulting RNA is reverse transcribed using RT primer 2, amplified by PCR, and analyzed by next-generation sequencing.
• FIG. 3 is a diagram that shows an early embodiment of the barcoding/parallelism strategy - showing the massively parallel RNA virus diagnostic assay.
• This diagram shows the workflow for a single diagnostic performed in parallel on thousands of samples.
• Step (1) Each BubblerTM contains a Unique barcode appended to the reverse transcription (RT) primer (drawn in purple), which is incorporated into the cDNA, as shown on the top and middle left. This copying event is the basis of diagnosis as it only occurs if the viral RNA is present in the sample.
• Step (4) The circles are amplified by inverted PCR primers and analyzed by next-generation sequencing. The bottom right describes the sequence analysis. The presence of barcodes (purple) is associated with a positive test result. The number of times a barcode is sequenced is proportional to the viral load. The (blue) copied viral sequences contain viral strain information useful in reconstructing transmission paths.
• FIG. 4 is a diagram showing a diagnostic matrix returned by an assay.
• Each kit contains an ensemble of uniquely barcoded RT primers specific to at least twenty-seven different RNAs. These RNAs are targeted to respiratory pathogens and human RNAs of varying abundance and various cellular origins.
• the assay identifies the pathogen and the quality of the sample.
• FIG. 5 shows a view of the BubblerTM device with several permutations for several uses.
• FIG. 5(A) For environmental sampling, a vacuum line is attached to the air vents to draw a continuous airflow through the BubblerTM.
• FIG. 5(B) The device can be miniaturized to a tube size compatible with liquid handling platforms.
• FIG. 5(C) Canola oil, mineral oil (any non-toxic oil).
• FIG. 5(D) The solution can be H20, TE solution, a readily substitutable replacement solution.
• DNAzolTM, RNAzolTM, phenol/chloroform 1 :1 solution, H20, TE solution can be used.
• FIG. 6 shows a molecular characterization of the bubbler's sample relative to alternate (tongue scrape, saliva) sampling technology.
• RT-PCR demonstrates the presence of cellular RNA (18S, bottom panel) but the absence of ACE2R (COVID-19 viral receptor). This finding supports the idea that the COVID-19 signal from the bubbler comes predominantly from VIRAL PARTICLES and not viral transcripts in infected cells
• FIG. 7 shows the implementation on a contrived COVID sample panel.
• the panel consists of ten serial 5-fold dilutions of a COVID standard (ATCC, VR-1986D, Lot 70035624) arrayed in a manner prescribed by the FDA emergency use authorization guidelines.
• Panel A shows the RT primer described in Figure 2.
• Panel B shows the dilution scheme used to calculate a detection limit of 334 viral particles /breath.
• the BubblerTM (which in Rhode Island and some other places can be called the BuhblahTM) is an attractive alternative to current swab-based sample collection technologies.
• the BubblerTM can replace the nasopharyngeal swab, stabilization, RNA extraction, and reverse transcription with a rapid, single step.
• This collection device samples aerosolized particles from human breath is a more direct and medically relevant sample for evaluating the risk of transmission than a nasopharyngeal swab.
• This handheld device captures aerosolized RNA containing particles from breath by bubbling through an oil/aqueous emulsion that contains enzymatic reverse transcriptase activity, which converts viral RNA into stable, molecularly barcoded cDNA.
• a key advance is including the RT step at the collection site as this enables the collection of samples compatible with large-scale downstream sequencing-based parallel diagnostics.
• Breathalyzers have been developed to sample metabolites. Prior studies failed to detect differences between the lung microbiome and the microbiome of the upper respiratory tract. See Charlson et al., Am. J. Respir. Crit. Care Med. (184), 957-963 (2011).
• ACE-2 ACE-2 expression is found but not restricted to the lung. Hermans &. Bernard, Am. J. Respir. Crit. Care Med. 159, 646-678 (1999).
• the invention provides a device and method orthogonal to existing
• COVID-19 testing protocols The parallelism could be expanded to include multiple tests or an entire respiratory panel in one BubblerTM so diseases with similar symptoms can be tested jointly.
• the device and method enable testing tens of thousands of people a day more conveniently and comfortably than taking a nasal swab.
• the rapid high-throughput assay enables large-scale survey sequencing.
• the BubblerTM can be dispatched for home use, decreasing the burden on current testing facilities.
• the invention described in this specification does not concern a process for cloning humans, processes for modifying the germ line genetic identity of humans, uses of human embryos for industrial or commercial purposes, or processes for modifying the genetic identity of animals likely to cause them suffering with no substantial medical benefit to man or animal, and also animals resulting from such processes.
• Air vent has the plain meaning of an opening that allows air to pass out of or into a confined space. Air vents that could contain airborne viruses are installed in hospital emergency rooms, airports, and building heating, ventilation, and air conditioning (HVAC) air handling systems.
• HVAC heating, ventilation, and air conditioning
• Airborne has the plain meaning of a particle that is traveling in the air.
• Aseptic Processing Facility is a building, or segregated segment of it, containing cleanrooms in which air supply, materials, and equipment are regulated to control microbial and particle contamination. See, United States Food and Drug Administration, Guidance for Industry, Sterile Drug Products Produced by Aseptic Processing — Current Good Manufacturing Practice (September 2004).
• “Clean area” or a “clean zone” is an area with defined particle and microbiological cleanliness standards. See United States Food and Drug Administration, Guidance for Industry, Sterile Drug Products Produced by Aseptic Processing — Current Good Manufacturing Practice (September 2004).
• Coronavirus has the biomedical art-recognized meaning of the group of related RNA viruses that cause diseases in mammals and birds.
• Coronaviruses constitute the subfamily Orthocoronavirinae, in the family Coronaviridae, order Nidovirales, and realm Riboviria. They are enveloped viruses with a positive-sense single-stranded RNA genome and a nucleocapsid of helical symmetry.
• COVID-19 (SARS-CoV-2) is a coronavirus that gains entry into a wide range of cell types through the ACE2 receptor and causes COVID-19, a severe respiratory disorder. COVID-19 is characterized by fever, a dry cough, and a variety of other symptoms. While COVID-19 can present with symptoms outside the lower respiratory tract, a dangerous trajectory can cause inflammation in the lungs resulting in pneumonia. Because SARS-CoV-2 is an airborne pathogen, the infection status of the lungs and airway is predictive not only of disease outcome but also the risk of transmission.
• DNA virus has the biomedical art-recognized definition of a virus whose genetic material is deoxyribonucleic acid. There are six generally-recognized classes of viruses. The DNA viruses constitute classes I (double stranded DNA viruses) and II (single-stranded DNA viruses).
• Environment has the plain meaning of the surroundings or conditions in which a person, animal, or plant lives or operates, especially the surrounding air.
• McNemar's test is a statistical test used on paired nominal data. It is applied to 2 c 2 contingency tables with a dichotomous trait, with matched pairs of subjects, to determine whether the row and column marginal frequencies are equal (that is, whether there is "marginal homogeneity").
• Nucleic acid and “nucleic acid molecule” can be used interchangeably herein and refer to a polymer or polymer block of nucleotides or nucleotide analogues.
• the nucleic acid may be obtained from natural sources or may be produced recombinantly or by chemical synthesis.
• the nucleic acid can be single, double, or multiple stranded and may comprise modified or unmodified nucleotides or nonnucleotides or various mixtures and combinations thereof.
• Patient to refer to any person to whom the assay is administered.
• a patient can refer to a person who has or is suspected of having COVID-19, to whom the assay is administered.
• patient can refer to a person who has or is suspected of having COVID-19, to whom the assay is administered.
• the terms “patient,” “individual,” and “subject” are interchangeable.
• PCR Polymerase chain reaction
• Common viral respiratory diseases are illnesses caused by a variety of viruses that have similar traits and affect the respiratory tract.
• the viruses involved may be the influenza viruses, respiratory syncytial virus (RSV) (the major cause of bronchiolitis, pneumonia, croup, bronchitis, and otitis media), parainfluenza viruses (the major cause of croup in young children and can cause bronchitis, pneumonia, and bronchiolitis), or respiratory adenoviruses (which can cause a variety of illnesses from pharyngitis to pneumonia, conjunctivitis, and diarrhea).
• Other viruses include rhinoviruses (which typically causes the common cold) and coronaviruses. Infection with viruses in the respiratory tract can cause complications such as tonsillitis, laryngitis, bronchitis, pneumonia. See Boncristiani, Respiratory viruses. Encyclopedia of Microbiology, 500-518 (February 17, 2009).
• Reverse transcriptase has the biomedical art-recognized meaning of an enzyme that catalyzes the formation of DNA from an RNA template in reverse transcription. Reverse transcriptase is commercially available.
• RNA Ribonucleic acid
• RNA has the biomedical art-recognized meaning of a ribose-containing nucleic acid. Its principal role is to act as a messenger carrying instructions from DNA for controlling the synthesis of proteins, although in some viruses RNA rather than DNA carries the genetic information.
• RNA virus has the biomedical art-recognized definition of a virus whose genetic material is ribonucleic acid.
• the RNA may be either double- or single-stranded.
• the DNA viruses constitute classes I and II.
• the RNA viruses make up the remaining classes.
• Class III viruses have a double-stranded RNA genome.
• Class IV viruses have a positive single-stranded RNA genome, the genome itself acting as mRNA (messenger RNA.
• Class V viruses have a negative single-stranded RNA genome used as a template for mRNA synthesis.
• Class VI viruses have a positive single- stranded RNA genome but with a DNA intermediate not only in replication but also in mRNA synthesis. Notable human respiratory diseases caused by RNA viruses include the common cold, influenza, SARS, MERS, and COVID- 19.
• Cochrane-Armitage test has the statistical art-recognized meaning.
• the Cochran-Armitage test for trend is used in categorical data analysis when the aim is to assess for the presence of an association between a variable with two categories and an ordinal variable with k categories. It modifies the Pearson chi-squared test to incorporate a suspected ordering in the effects of the k categories of the second variable.
• the BubblerTM is a hand-held device with a glass straw at the top into which the subject being tested blows a breath.
• the glass straw can be made from a Pasteur pipette.
• the device is a breathalyzer for viruses.
• This device can be used to determine who is infected with a respiratory virus, such as an RNA virus, such as an influenza virus, rhinovirus, or coronavirus, such as COVID-19. See FIG. 4.
• the device enables a person having ordinary skill in the medical art to test tens of thousands of people a day so it is easier, more convenient, and more comfortable than taking a nasal swab.
• Step (1) Hold the BubblerTM from the top. See FIG. 1 (B).
• Step (2) Tilt slightly down and take a normal breath.
• Step (3) Exhale into the tube.
• the person being tested or the person performing the test should hear a bubbling sound.
• the person being tested shall blow out all the air in the lungs. This process should take no more than 10 seconds.
• the oil/water mix at the bottom of the tube should be an emulsion (like oil and vinegar salad dressing). Sometimes a little saliva gets into the BubblerTM, so the last step is to get a saliva sample.
• the real-time PCR program is set as: (1) hold stage: 50°C for two minutes, then 95°C for three minutes; (2) PCR: 95°C for fifteen seconds, 60°C for twenty seconds and 72°C for thirty seconds, 40 cycles; 3). Melt curve: 95°C at fifteen seconds, 60°C for twenty seconds, then increase to 95°C with the speed of 0.05°C/s, hold at 95°C for fifteen seconds.
• Ct ⁇ 35 for both real-time PCR primer sets the patient sample is determined as positive for Sars-Cov-2.
• GoTaq Master Mix Promega, M7123
• Human total RNA Thermo Fisher, 4307281 , Lot 00890901
• SARS-CoV-2 genomic RNA ATCC, VR-1986D, Lot 70035624 are used as controls.
• RNA used in human-constructed samples was quantified by qPCR. [0070] RNA was reverse transcribed to cDNA via SuperscriptTM IV transcriptase
• a POS BubblerTM result are BubblersTM that were POS from patients that were also POS on either of the duplicated L-PCR assays.
• a NEG BubblerTM result are BubblersTM that were NEG from patients that were also NEG on either of the duplicated L-PCR assays.
• RNA transcription DNA oligonucleotide of SARS-CoV-2 N gene with a T7 promoter was synthesized at Integrated DNA Technologies (IDT). PCR amplification was performed on this oligonucleotide using Q5 High fidelity DNA polymerase (NEB) to prepare a template for in vitro transcription (IVT). Primers were listed in Table S1. A single PCR amplicon was confirmed by agarose gel electrophoresis. IVT was performed using Riboprobe® System-T7 kit (Promega, Cat# P1440) following the manufacture's recommendations. The DNA template was removed by digestion with DNase i, and IVT RNA was subsequently extracted using phenol (pH4.7):chloroform and precipitated by ethanol.
• the expected read count for a given dilution level is set to one fifth the number of reads observed from the previous level.
• the expectation for the read counts of the two water-based blank samples was taken to be zero.
• the expected read count for the first dilution level was set to the observed read count for plotting purposes, but this level was excluded from correlation calculations.
• the Pearson correlation coefficient was calculated for these comparisons: observed FP1 counts vs. observed FP2 counts, observed FP1 counts vs. expected FP1 counts, and observed FP2 counts vs. expected FP2 counts.
• DNA oligonucleotide of SARS-CoV-2 N gene with a T7 promoter was synthesized at Integrated DNA Technologies (IDT). PCR amplification was performed on this oligonucleotide using Q5 High fidelity DNA polymerase (NEB) to prepare a template for in vitro transcription (IVT). Primers are listed in the SEQUENCE LISTING. A single PCR amplicon was confirmed by agarose gel electrophoresis.
• RNA template was removed by digestion with DNase L and transcribed RNA was subsequently extracted using phenol (pH4.7):chloroform and precipitated by ethanol.
• Each barcoded primer contains a targeting region either binding to human 18S rRNA or N gene, a 3-nucleotide randomer (Unique Molecular Identifiers, UMI), a 8-nucleotide barcode, a constant region where PCR reverse primer binds and a T7 promoter. See the SEQUENCE LISTING for primers. Thirty-six human- constructed samples were arrayed into 96-well where barcoded RT primers were already assigned and each well contained 2-barcoded RT primers, one for 18S rRNA and the other for N gene RNA.
• UMI Unique Molecular Identifiers
• QIAquick PCR Purification kit Qiagen, Cat #28004
• the following two step nested PCR amplification uses the same reverse primer and two different forward primers.
• Specific RT and PCR primers were listed in the SEQUENCE LISTING. Amplicon sequencing was performed to quantify each barcode.
• RT primers hRPp1_RT - NNNNNNGAATTGGGTTA SEQ ID NO: 1.
• Real-time PCR primers hRPp1_rtPCR_1 - GGATGCCTCCTTTGCCGGAG (SEQ ID NO: 14).
• Cv19N_rtPCR1_1 - AGTCAAGCCTCTTCTCGTTCC SEQ ID NO: 16
• Cv19N_rtPCR1_2 - G C A AAG CAAGAGCAGCATCAC (SEQ ID NO: 17).
• RT primer 1 in FIG. 2 refers to from N-aene-BC1 to N-aene-BC36.
• N-gene-BC1 - TAATACGACTCACTATAGGGCCGATATCCGACGGTAGTGTCACGTCG TN N NAT CAT CCAAAT CT G CAG (SEQ ID NO: 20).
• N-gene-BC2 - TAATACGACTCACTATAGGGCCGATATCCGACGGTAGTGTCAATTGA TN N NAT CAT CCAAAT CT G CAG (SEQ ID NO: 21).
• N-gene-BC3 - TAATACGACTCACTATAGGGCCGATATCCGACGGTAGTGTATATTGTA NNNATCATCCAAATCTGCAG (SEQ ID NO: 22).
• N-gene-BC5 - TAATACGACTCACTATAGGGCCGATATCCGACGGTAGTGTACACATG TN N NAT CAT CCAAAT CT G CAG (SEQ ID NO: 24).
• N-gene-BC8 - TAATACGACTCACTATAGGGCCGATATCCGACGGTAGTGTATGCTTG AN N NAT CATCCAAAT CTG CAG (SEQ ID NO: 27).
• N-gene-BC10 - TAATACGACTCACTATAGGGCCGATATCCGACGGTAGTGTCAGGCA TTN N N AT CAT C C AAAT CTG CAG (SEQ ID NO: 29).
• N-gene-BC13 AATACGACTCACTATAGGGCCGATATCCGACGGTAGTGTGAACGAC AN N NAT CATCCAAAT CTG CAG (SEQ ID NO: 32).
• N-gene-BC15 - TAATACGACTCACTATAGGGCCGATATCCGACGGTAGTGTGTAACC GAN N NAT CAT CCAAAT CT G CAG (SEQ ID NO: 34).
• N-gene-BC17 - TAATACGACTCACTATAGGGCCGATATCCGACGGTAGTGTGACACT TANNNATCATCCAAATCTGCAG SEQ ID NO: 36.
• N-gene-BC18 - TAATACGACTCACTATAGGGCCGATATCCGACGGTAGTGTGGTTGG AC N N N AT CAT CC AAAT CTG C AG SEQ ID NO: 37.
• N-gene-BC20 - TAATACGACTCACTATAGGGCCGATATCCGACGGTAGTGTTATGCC AG
• N NAT CAT CCAAAT CT G CAG (SEQ ID NO: 39).
• N-gene-BC23 - TAATACGACTCACTATAGGGCCGATATCCGACGGTAGTGTTGTATG CG N N NAT CATCCAAAT CT GCAG (SEQ ID NO: 42).
• N-gene-BC24 - TAATACGACTCACTATAGGGCCGATATCCGACGGTAGTGTTCCAGT CG N N NAT CATCCAAAT CT GCAG (SEQ ID NO: 43).
• N-gene-BC30 - TAATACGACTCACTATAGGGCCGATATCCGACGGTAGTGTTGTGGT TG N N N AT CAT CCAAAT CTG CAG (SEQ ID NO: 49).
• N-gene-BC33 - TAATACGACTCACTATAGGGCCGATATCCGACGGTAGTGTTGCGAT CTN N NAT CAT CCAAAT CTG CAG (SEQ ID NO: 52).
• N-gene-BC34 - TAATACGACTCACTATAGGGCCGATATCCGACGGTAGTGTTGCATA GTNNNATCATCCAAATCTGCAG (SEQ ID NO: 53).
• N-gene-BC35 TAATACGACTCACTATAGGGCCGATATCCGACGGTAGTGTTGATAC GTNNNATCATCCAAATCTGCAG (SEQ ID NO: 54).
• N-gene-BC36 - TAATACGACTCACTATAGGGCCGATATCCGACGGTAGTGTTCGAGC GTNNNATCATCCAAATCTGCAG (SEQ ID NO: 55).
• 18S-rRNA-BC2 - TAATACGACTCACTATAGGGCCGATATCCGACGGTAGTGTCGATG TTTNNNGACGGGCGGTGTGTAC (SEQ ID NO: 57).
• 18S-rRNA-BC3 - TAATACGACTCACTATAGGGCCGATATCCGACGGTAGTGTTTAGG CATNNNGACGGGCGGTGTGTAC (SEQ ID NO: 58).
• 18S-rRNA-BC4 - TAATACGACTCACTATAGGGCCGATATCCGACGGTAGTGTACAGT GGTNNNGACGGGCGGTGTGTAC (SEQ ID NO: 59).
• 18S-rRNA-BC5 - TAATACGACTCACTATAGGGCCGATATCCGACGGTAGTGTGCCAA TGTNNNGACGGGCGGTGTGTAC (SEQ ID NO: 60).
• 18S-rRNA-BC6 - TAATACGACTCACTATAGGGCCGATATCCGACGGTAGTGTCAGAT CTGNNNGACGGGCGGTGTGTAC (SEQ ID NO: 61).
• 18S-rRNA-BC9 - TAATACGACTCACTATAGGGCCGATATCCGACGGTAGTGTTGGTT GTTNNNGACGGGCGGTGTGTAC (SEQ ID NO: 64).
• 18S-rRNA-BC10 - TAATACGACTCACTATAGGGCCGATATCCGACGGTAGTGTTGTAC CTTNNNGACGGGCGGTGTGTAC (SEQ ID NO: 65).
• 18S-rRNA-BC15 - TAATACGACTCACTATAGGGCCGATATCCGACGGTAGTGTTGCAT AGTNNNGACGGGCGGTGTGTAC (SEQ ID NO: 70).
• 18S-rRNA-BC20 - TAATACGACTCACTATAGGGCCGATATCCGACGGTAGTGTTTCCA TTGNNNGACGGGCGGTGTGTAC (SEQ ID NO: 75).
• 18S-rRNA-BC24 - TAATACGACTCACTATAGGGCCGATATCCGACGGTAGTGTTACTT CGGNNNGACGGGCGGTGTGTAC (SEQ ID NO: 79).
• 18S-rRNA-BC30 - TAATACGACTCACTATAGGGCCGATATCCGACGGTAGTGTTCAG ATTCNNNGACGGGCGGTGTGTAC (SEQ ID NO: 85).
• 18S-rRNA-BC35 - TAATACGACTCACTATAGGGCCGATATCCGACGGTAGTGTGGCA AGCANNNGACGGGCGGTGTGTAC (SEQ ID NO: 90).
• 18S-rRNA-BC36 - TAATACGACTCACTATAGGGCCGATATCCGACGGTAGTGTGAAC GACANNNGACGGGCGGTGTGTAC (SEQ ID NO: 91).
• RT primer 2 for 18S rRNA - GATTTGTCTGGTTAATTCCGATAACG (SEQ ID NO: 92).
• RT primer 2 for N gene - CGTGGTCCAGAACAAACCCA SEQ ID NO: 93).
• the RT primer 2 in FIG. 2 refers to RT primer 2 for N gene.
• the FP1 in FIG. 2 refers to FP1 for N gene.
• FP2 for N gene - CTGAATAAGCATATTGACGCATAC (SEQ ID NO: 98).
• the FP2 in FIG. 2 refers to FP2 for N gene.
• RP- CCGATATCCGACGGTAGTGT (SEQ ID NO: 99).
• the RP in FIG. 2 refers to RP for N gene.
• IVT-PCR-FP - GTAAAACGACGGCCAGTGAATT SEQ ID NO: 100.
• IVT-PCR-RP - CAGGAAACAGCTATGACCATG SEQ ID NO: 101.
• RNA virus particles from human breath were readily precipitated from the interior surface of an inflated party balloon after a one-hour incubation at -20°C. rRNA can be readily detected by RT-PCR in this liquid with no RNA extraction. This collection technique was simple.
• This EXAMPLE shows a test of the efficacy of a device and method of sample collection.
• the inventors developed a successful prototype that can copy 18S rRNA from breath with the same efficiency as conventionally sampled RNA.
• the inventors (A) applied the BubblerTM in parallel with conventional nasopharyngeal swab testing for comparison, not for a diagnostic purpose; and (B) characterized the BubblerTM patient isolate using standard molecular biological diagnostics (Western, PCR, sequencing). The collected sample was evaluated for lung epithelial markers, potential saliva contamination, and viral RNA to determine the most abundant viral regions for improved amplicon design.
• the standard of care test for this population is a NP-PCR-based assay, which the patients received besides the investigational test and was used as the gold standard for comparison.
• BubblerTM test this includes the standard of care NP-PCR test performed during their visit and any other charges associated with their visit.
• Inclusion criteria The patients recruited for the clinical study were 18 years and older who presented with symptoms consistent with undiagnosed COVID infection. These patients had already undergone a standard of care nasopharyngeal swab and were waiting for their results or would have already received a positive result. [0088] Exclusion criteria: Patients with asthma or COPD exacerbated by COVID infection were excluded from the clinical study because they could not sustain an exhalation into the BubblerTM. Patients who had burns or trauma to the mouth were also excluded. Patients who unable to provide signed consent were excluded.
• Study protocol The patients were first screened for entry into the clinical study. The patients were then shown how to use the BubblerTM using a different exemplar device during exhalation to a study participant. This demonstration showed the patient what to avoid, such as accidental inhalation. The patients were observed they used the BubblerTM. Upon entry to the clinical study, the patients were offered the opportunity to sign a Specimen Banking form, enabling the storage of what is left of the sample after analysis. Using hospital records, the patient’s course was followed by noting demographics, historical and physical exam information, vital signs, laboratory findings, length of stay, mortality, and results of infection-related diagnostics and interventions, and pulse oximeter readings.
• Standardized treatment The clinical study was discussed with the patients who consented at that time. This clinical study was a prospective observational trial. Data from this clinical study was not available to patient providers at the time of provision of care and was not used to influence care or disposition of patients.
• Primary outcome The inventors tested the BubblerTM device in parallel with conventional nasopharyngeal swab testing. This testing was for comparison, not for a diagnostic purpose.
• the inventors characterized the BubblerTM patient isolates using standard molecular biological diagnostics known to persons having ordinary skill in the biomedical art (Western analysis, PCR, sequencing). The collected sample was evaluated for lung epithelial markers, potential saliva contamination, and viral RNA to determine the most abundant viral regions for improved amplicon design.
• the devices used in the clinical study were made using a sterile construction protocol, are kept sterile until used, and pose little risk of patient contamination.
• the device was constructed by people wearing gloved hands, sprayed with 70% ethanol and allowed to dry overnight in an ultraviolet (UV) hood. Emulsion is added before use under a sterile protocol.
• UV ultraviolet
• Data collected on each enrolled patient included name, gender, age, medical record number, account identifier, birth date, admission date, pertinent admission conditions, hemodynamics, therapeutic interventions, diagnostics (e.g., culture results, radiography results, blood analysis, pathology summaries), disposition status and hospital date/day. These data could verify the diagnosis of COVID-related infection, to score illness severity, and structure a demographically appropriate control in the event one is needed. Again, all identifying information on each patient was be kept separate from clinical study samples. Data used for analysis did not include patient name, MR number, account identifier, birth date, admission date, or disposition date.
• COPD Chronic Obstruction Pulmonary Disease
• Time of Blood Collection (24 hr format): : _
• Radiography Results _ [00140] NOTE: Please print and attach a copy of the participant’s Chest X-ray interpretation.
• the inventors improved SARS-CoV-2 detection by simplifying the assay and broadening the compartments tested. Then, the inventors designed a clinical study to sample COVID from three points in the respiratory tract. Oral samples by saliva/tongue scrapes or exhaled breath were compared to the traditional nasopharyngeal swab. To simplify the assay, the inventors explored the viability of performing reverse transcription directly on a sample without RNA extraction, eliminating the need to stabilize a sample and allowing the assay to be performed at home the inventors describe the design and testing of a breathalyzer called the BubblerTM that directly samples aerosolized particles in exhaled breath.
• BubblerTM a breathalyzer
• BubblerTM samples had a similar level of RT-PCR efficiency to RNA extracted from cultured cells. More rRNA could be detected from a single (less than ten seconds) breath than could be detected from conventionally-extracted RNA.
• the inventors optimized the device and demonstrated that the BubblerTM could be miniaturized and the RT reaction mixture was stable in the kit for at least two weeks. See FIG. 5.
• kits were constructed to include one BubblerTM and two saliva/tongue scrapes as controls.
• Several experiments were conducted to compare samples collected from the BubblerTM to the control. Interestingly, samples collected from the tongue scrape were positive for expression of the ACE2 receptor whereas ACE2 signal was undetectable in BubblerTM samples, suggesting the BubblerTM and tongue scrape sample RNA from distinct compartments. See FIG. 6.
• RT-PCR assay to amplify SARS-CoV-2 RNA was optimized on a commercially available positive control. The optimization yielded RT and PCR primers that performed with similar sensitivity to the CDC primers, N1 and N2. See FIG. 2. Amplification of the housekeeping gene RNase P was used as a sample control. Reverse transcriptase reaction mixtures were added to BubblersTM and sample tubes and packaged in test kits administered to consenting enrolled patients during their treatment at Rhode Island Hospital. A total of seventy patients were tested over a period of approximately 7 months. See Figure 3.
• the H-PCR showed a PPV of 0.95 for abnormal chest X-rays (positive XR).
• the H-PCR showed a PPV of 0.69 for confirmed positive BubblerTM tests.
• the confirmed positive BubblerTM tests showed a PPV of 0.94 for positive XRs.
• the L-PCR confirmed BubblerTM results showed equally strong prediction for a positive XR as the H-PCR positive results.
• RNase P is expected to be expressed in every cell, whereas SARS-CoV-2 RNA is presumably localized to airborne viral particles and material released from lysed cells.
• the data obtained in this EXAMPLE showed the BubblerTM sample is more weighted towards viral particles as the ratio of CT scores of SARS-CoV-2 to RNase P were over 3-fold higher than observed in the tongue scrape.
• FIG. 7(A) An advantage of performing reverse transcription in the collection tube was to use barcoded cDNA in a high throughput testing scheme.
• FIG. 7(A) Each RT primer targets a window of RNA but still functions with an additional sequence at the 5' end. This sequence consisted of a T7 promoter to amplify the signal, a 6-nucleotide sample barcode, and a 3-nucleotide random tag to distinguish unique RT events from duplicates that arise in amplification.
• barcoded primers were used to test in triplicate a series of ten five-fold dilutions of SARS-CoV-2 and two water-based blanks.
• Samples are reverse transcribed, pooled and then subjected to a two-step nested PCR strategy. See FIG. 7(B).
• barcodes were counted and associated with individual amplification events. Barcode counts were highly correlated across replicates and with the expected counts. The correlation was lost at the 5 th serial dilution corresponding to a detection limit of 334 genomic copies.
• BubblerTM matching the hospital assay in predicting abnormal X-ray results
• these results show that the BubblerTM samples a compartment enriched in SARS-CoV-2 virus which is likely to be a better indicator of current infection than nasopharyngeal swabs.
• the United States Centers for Disease Control recommends upper respiratory specimens for initial diagnostic testing for SARS-CoV-2 infection. Despite yielding the highest viral loads for the detection of SARS-CoV-2, sample collection by sputum induction is not recommended due to the likelihood of aerosolization. Collection of lower respiratory tract samples from patients with suspected COVID-19 pneumonia is only recommended when an upper respiratory tract sample is negative. See the United States National Institutes of Health Coronavirus Disease 2019 (COVID-19) Treatment Guidelines.
• nasopharyngeal swab The most common testing for upper respiratory specimens has been the nasopharyngeal swab.
• nasopharyngeal swabs also carry an aerosolization risk as they are so uncomfortable that patients often cough, sneeze or gag during the procedure, e.g., one patient refused conventional swab.
• Alternative assays such as the BubblerTM estimate lower respiratory samples, with the safety of an upper respiratory sample.
• finding nasopharyngeal swab alternatives can relieve supply chains for the swabs and transport media, reduce the need for personal protective equipment during aerosolization and provide a more comfortable patient experience.
• Testing strategies for active or prior infection rely on detection of viral RNA or antibodies to the virus. Collection is usually performed in the upper respiratory tract by saliva or nasopharyngeal swab, which have comparable sensitivities (97% agreement).

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